Enterococcus faecalis V583 Contains a Cytochrome bd-Type Respiratory Oxidase

ABSTRACT We have cloned an Enterococcus faecalis gene cluster,cydABCD, which when expressed in Bacillus subtilis results in a functional cytochrome bdterminal oxidase. Our results indicate that E. faecalisV583 cells have the capacity of aerobic respiration when grown in the presence of heme.

after inoculation, cells were harvested by centrifugation and washed in 20 mM sodium MOPS buffer, pH 7.4. All subsequent steps were done at 4°C or on ice. Cells were suspended in MOPS buffer containing DNase (0.1 mg/ml) (bovine pancreas, type 1; Sigma), 0.5 mM phenylmethylsulfonylfluoride and 5 mM MgSO 4 and broken using a French pressure cell. After a centrifugation at 5,000 ϫ g, for 15 min, membranes were harvested from the supernatant by centrifugation at 200,000 ϫ g, for 90 min, washed once, and then suspended in MOPS buffer. Protein concentrations were determined using the bicinchoninic acid assay (Pierce) with bovine serum albumin as the standard. Light absorption spectra were recorded as described previously (28).
Isolated membranes from E. faecalis cells grown aerobically in the presence of hemin demonstrated light absorption difference spectra with features characteristic for cytochrome bd, with absorption peaks at 561 nm (cytochrome b) and 626 nm (cytochrome d) (Fig. 1, spectrum A). The trough at about 650 nm indicates the presence of a stable oxygenated cytochrome d species [Fe(II)-O 2 ] (19). Membranes from cells grown without hemin lacked spectroscopically detectable cytochromes (Fig. 1,

spectrum B).
A cyd gene cluster is present in E. faecalis. The amino acid sequence of B. subtilis CydA was used to search for related sequences in the preliminary release of the E. faecalis genomic data obtained from The Institute for Genomic Research (TIGR). The BLAST search (1) resulted in the identification of a contig containing four putative genes, similar to B. subtilis cydA, cydB, cydC, and cydD. Alignments of the B. subtilis and E. faecalis amino acid sequences showed a sequence identity of 56% for CydA, 46% for CydB, 51% for CydC, and 49% for CydD.
To clone the E. faecalis cydABCD genes, the DNA sequence obtained from the TIGR E. faecalis database was used to design two primers, ECYD1 (5Ј GGAGATCTAATGGAAAT GAACAATTCAGGTAAG-3Ј) (BglII restriction site underlined) and ECYD2 (5Ј-GGTCTAGACTATCATGGCGTTAC AGAAGCAC-3Ј) (XbaI restriction site underlined). The BglII restriction site is located 59 nucleotides upstream of the putative translational initiation site of cydA. These primers were used in a long-range PCR (Expand High Fidelity PCR system; Roche) with 500 ng of E. faecalis chromosomal DNA (prepared essentially as described by Hoch [9]) as the template. The amplified 6.2-kb fragment was cut with restriction enzymes BglII and XbaI and ligated into plasmid pCYD26, cut with BamHI and XbaI. Plasmid pCYD26 contains the B. subtilis cyd promoter region (nucleotides Ϫ192 to ϩ199 with respect to the transcription start site) (28) in the low-copy-number vector pHPSK. The ligate was used to transform B. subtilis 168A to chloramphenicol resistance, resulting in plasmid pLUF04 (Fig.  2). Restriction site mapping and partial DNA sequence analysis confirmed the identity of the cloned fragment.
Expression of E. faecalis cydABCD in B. subtilis. To facilitate characterization of E. faecalis cytochrome bd, we aimed to find a method for overproduction of the enzyme complex. Since cytochrome bd from B. subtilis and E. faecalis appeared to be closely related, we choose to use B. subtilis as our expression host. Strains and plasmids used in this study are listed in Table  1. B. subtilis strain LUW20 lacks cytochrome bd, and hence membranes from this strain lack the spectroscopic features of cytochrome bd (28). LUW20/pLUF04 (E. faecalis cydABCD), LUW20/pCYD23 (B. subtilis cydABCD) (28), and LUW20/ pCYD26 (vector only) were grown at 37°C in nutrient sporulation medium with phosphate (4) supplemented with 0.5% glucose (NSMPG) and chloramphenicol (5 mg/liter). The cultures were harvested in the stationary phase. Membranes were prepared as described previously (7) and suspended in 20 mM sodium MOPS buffer, pH 7.4. Light absorption difference spectra of membranes from LUW20/pLUF04 showed an increased absorption at 561 nm and a peak at 626 nm, due to expression of a cytochrome bd (Fig. 3, spectrum B). Membranes from LUW20/pCYD23 showed a spectrum with an increased absorption at 563 nm and a peak at about 627 nm (Fig. 3, spectrum C), whereas membranes from the control, LUW20/pCYD26, lacked the peaks characteristic for cytochrome bd (Fig. 3, spectrum A), as expected. The absorption peak at about 600 nm in the spectra is mainly due to cytochrome a of the cytochrome aa 3 oxidase (27)  aa 3 , but carrying pLUF04, could grow under aerobic conditions. Chromosomal DNA from LUH14 (⌬qoxABCD::kan), prepared as described by Hoch (9), was used to transform LUW20/pLUF04, LUW20/pCYD23, and LUW20/pCYD26 to kanamycin resistance. The same limiting amount of LUH14 DNA (0.2 mg/liter of competent cells) was used for all three strains. Transformants were selected on tryptose blood agar base plates supplemented with 1% (wt/vol) glucose and containing chloramphenicol (5 mg/liter) and kanamycin (5 mg/ liter). To verify that the transformants obtained still lacked the chromosomal copy of the B. subtilis cydABCD operon, they were streaked on plates containing tetracycline (15 mg/liter). As shown in Table 2, kanamycin-and tetracyclineresistant transformants, i.e., transformants deleted for both the qoxABCD and the cydABCD operons in the B. subtilis chromosome, were obtained only with LUW20/pLUF04 and LUW20/ pCYD23. One transformant from each strain was kept and designated LUW174 and LUW173, respectively. The few transformants obtained with LUW20/pCYD26 were all sensitive to tetracycline; i.e., the tetracycline resistance marker in LUW20 had been substituted with the B. subtilis cydABCD operon from the LUH14 chromosomal DNA. To further characterize LUW174 and LUW173 and to compare the spectroscopic features of E. faecalis V583 cytochrome bd and B. subtilis cytochrome bd in more detail, the two strains were grown in NSMPG and membranes were prepared as described above. The growth properties of LUW174 did not differ from those of LUW173. Light absorption difference spectra of membranes from LUW174 (containing E. faecalis cytochrome bd) showed peaks at about 561, 595, and 626 nm (Fig. 4, spectrum A), indicating the presence of three prosthetic groups (low-spin heme b, high-spin heme b, and heme d). Membranes from LUW173 (containing B. subtilis cytochrome bd) showed peaks at about 563, 597, and 627 nm (Fig.  4, spectrum B). The absence of a peak at about 600 nm confirmed that the strains lack cytochrome aa 3 .
Conclusion. In this work, we show that E. faecalis V583 contains a cydABCD gene cluster and that membranes from this strain grown in the presence of heme contain a cytochrome bd. Under these growth conditions, cytochrome bd is the major (and possibly the only) membrane-bound cytochrome in E. faecalis. The cloned E. faecalis cydABCD gene cluster expressed in B. subtilis resulted in a cytochrome bd which showed a spectrum indistinguishable from that of the cytochrome bd  3 0 a B. subtilis LUW20, carrying a ⌬cydABCD::tet mutation in the chromosome and one of three plasmids, was transformed with chromosomal DNA from LUH14, carrying a ⌬qoxABCD::kan mutation. All strains showed a similar degree of competence as tested by transformation with plasmid pDG148.
b Transformants were selected on plates containing kanamycin and chloramphenicol and then restreaked on plates containing tetracycline. c Km r and Tet r indicate resistance to kanamycin and tetracycline, respectively.
found in E. faecalis. The E. faecalis cytochrome bd can functionally complement a B. subtilis cytochrome bd-deficient mutant, indicating that the E. faecalis cytochrome bd is a menaquinol oxidase. E. faecalis and B. subtilis both contain naphthoquinones in the cytoplasmic membrane: demethylmenaquinone and menaquinone, respectively (2). Thus, a specific electron donor for cytochrome bd is present in E. faecalis.
These results indicate that E. faecalis is capable of aerobic respiration. The physiological importance of the identified respiratory system and its role in pathogenesis remain to be determined.